Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast

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Abstract

Biosensors are valuable and versatile tools in synthetic biology that are used to modulate gene expression in response to a wide range of stimuli. Ligand responsive transcription factors are a class of biosensor that can be used to couple intracellular metabolite concentration with gene expression to enable dynamic regulation and high-throughput metabolite producer screening. We have established the Saccharomyces cerevisiae WAR1 transcriptional regulator and PDR12 promoter as an organic acid biosensor that can be used to detect varying levels of para-hydroxybenzoic acid (PHBA) production from the shikimate pathway and output green fluorescent protein (GFP) expression in response. The dynamic range of GFP expression in response to PHBA was dramatically increased by engineering positive-feedback expression of the WAR1 transcriptional regulator from its target PDR12 promoter. In addition, the noise in GFP expression at the population-level was controlled by normalising GFP fluorescence to constitutively expressed mCherry fluorescence within each cell. These biosensor modifications increased the high-throughput screening efficiency of yeast cells engineered to produce PHBA by 5,000-fold, enabling accurate fluorescence activated cell sorting isolation of producer cells that were mixed at a ratio of 1 in 10,000 with non-producers. Positive-feedback, ratiometric transcriptional regulator expression is likely applicable to many other transcription-factor/promoter pairs used in synthetic biology and metabolic engineering for both dynamic regulation and high-throughput screening applications.
LanguageEnglish
Article number1
Pages1-13
Number of pages13
JournalSynthetic Biology
Volume2
Issue number1
DOIs
Publication statusPublished - 29 Jan 2017

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Biosensing Techniques
Green Fluorescent Proteins
Yeasts
Synthetic Biology
Transcription Factors
Fluorescence
Metabolic Engineering
Gene Expression
Cell Separation
Saccharomyces cerevisiae
Noise
Flow Cytometry
Ligands
Acids
Population
4-hydroxybenzoic acid

Bibliographical note

Copyright the Author(s) 2017. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

Keywords

  • biosensor
  • adaptive laboratory evolution
  • synthetic biology
  • metabolic engineering
  • yeast

Cite this

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